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Zusammenfassung: Die mikrobiologische Grundlagenforschung benötigt geeignete Modellsysteme, um infektionsbiologische Fragestellungen in sinnvoller Weise bearbeiten zu können. Zum gegenwärtigen Zeitpunkt erfolgen solche Infektionsstudien meist im Rahmen von Tierversuchen, was aus ethischen Gründen aber umstritten ist. Eine mögliche Alternative, gerade für lokalisierte Infektionen, bieten Modelle auf der Basis von in vitro rekonstituiertem humanem Epithel oder rekonstituierter humaner Epidermis (RHE). In den letzten Jahren wurden diese Modellsysteme erfolgreich zur Evaluierung der Wirksamkeit topischer Antiinfektiva, zur Charakterisierung von fungalen Virulenzfaktoren und zur Beschreibung der Immunantwort bei lokalisierten Candida-albicans-Infektionen eingesetzt. Erst kürzlich wurden diese Modelle um weitere infektionsrelevante Immunzellen wie Lymphozyten, polymorphkernige Leukozyten, Mastzellen oder dendritische Zellen erweitert um deren Einfluss auf den Infektionslauf und die Interaktion zwischen Hautbarrieren und akzessorischen Immunzellen zu charakterisieren. Obgleich für RHE-Modelle die größte Erfahrung bei Candida-albicans-Infektionen besteht, können solche Modellsysteme auch für Infektionen mit anderen Pilzen oder auch Bakterien eingesetzt werden.

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

ABSTRACTCandida glabratahas emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabratastrains for in vitroand in vivostudies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitrogrowth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabratais capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabratahistidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivostudies. Notably, our work indicates that C. glabratahas successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

Candida species, in particular C. albicans, represent a major threat to immunocompromised patients. Able to exist as a commensal on mucosal surfaces of healthy individuals, these opportunistic fungi frequently cause superficial infections of mucosae and skin. Furthermore, in hospital settings, Candida species may cause life-threatening invasive infections in a growing population of vulnerable patients. In fact, candidaemia is associated with the highest crude mortality of all bloodstream infections. Candida cells may enter the bloodstream by direct penetration from epithelial tissues, due to damage of barriers in the body caused by surgery, polytrauma or drug treatment, or may spread from biofilms produced on medical devices. From the bloodstream, cells may infect almost all organs but appear to prefer certain organs depending upon the route of infection. The exact mechanisms by which Candida cells survive the challenge of the blood environment and escape from the bloodstream to cause deep-seated infections have not yet been elucidated, but various investigations are reviewed. It is clear, however, that Candida must have particular attributes which enable the organism to survive and grow within the environment of healthy individuals and to invade tissues in the immunocompromised host. Most studies have focussed on C. albicans and this review will therefore summarise work on the various known virulence factors and methods used to identify further virulence attributes of this fungus.

Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis. To define potential virulence factors, we investigated 50 strains of C. albicans in a murine peritonitis model. There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice. Alanine aminotransferase (ALT) and alpha-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively. The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro. When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities. This indicates that proteinases contributed to virulence in this model. Using strains of C. albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, or SAP4 through SAP6 (collectively referred to as SAP4-6), we showed that only a Deltasap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Deltasap1, Deltasap2, and Deltasap3 mutants, the ALT induced by the Deltasap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.

Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multigene family with at least nine members (SAP1 to SAP9) and are considered putative virulence factors important for the pathogenicity of this human pathogen. The role of Sap isoenzymes in the virulence of C. albicans has not yet been clearly established, and therefore, using recent progress in the genetics of this yeast, we have constructed a panel of isogenic yeasts, each with a disruption of one or several SAP genes. We focused on the construction of a C. albicans strain in which three related SAP genes (SAP4, SAP5, and SAP6) were disrupted. Growth of the delta sap4,5,6 triple homozygous null mutant DSY459 in complex medium was not affected, whereas, interestingly, growth in a medium containing protein as the sole nitrogen source was severely impaired compared to the growth of the wild-type parent strain SC5314. Since the presence of Sap2 is required for optimal growth on such medium, this suggests that Sap4, Sap5, or Sap6 plays an important role for the process of induction of SAP2. When guinea pigs and mice were injected intravenously with DSY459, their survival time was significantly longer than that of control animals infected with the wild-type SC5314. Attenuated virulence of DSY459 was followed by a significant reduction of yeast cells in infected organs. These data suggest that the group of Sap4, Sap5, and Sap6 isoenzymes is important for the normal progression of systemic infection by C. albicans in animals.

Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.

The gene encoding the Candida albicansaspartate proteinase that is secreted by cells grown in protein-containing media was cloned from a C. albicansgenomic bank. The base sequence of the insert shows a 1173 bp open-reading frame and indicates an amino acid sequence typical of aspartate proteinases, with amino acid sequence homology to other enzymes of this class and a putative signal peptide consisting of 50 amino acids upstream of the active enzyme.

The aspartate proteinase inhibitor pepstatin A has been shown previously to reduce the adherence of Candida albicansyeast cells to human surfaces. This suggests that in addition to their presumed function facilitating tissue penetration, the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles as cellular adhesins. We therefore examined the relative adherence of yeast cells of a parental wild‐type strain of C. albicansin relation to yeast cells of strains harbouring specific disruptions in various members of the SAP gene family in an otherwise isogenic background. The adhesiveness of Δsap1, Δsap2and Δsap3null mutants and a triple Δsap 4–6disruptant was examined on three surfaces – glass coated with poly‐l‐lysine or a commercial cell‐free basement membrane preparation (Matrigel) and on human buccal epithelial cells. Pepstatin A reduced adherence to all surfaces. Adherence of the each of the single SAP null mutants to these three substrates was either reduced or not affected significantly compared to that of the parental strain. The adherence of the Δsap4–6mutant was reduced on poly‐l‐lysine and Matrigel, but increased on buccal cells. The results suggest that in addition to a primary enzymatic role, various SAPs may also act singly or synergistically to enhance the adhesiveness to C. albicanscells to certain human tissues.

The secreted aspartyl proteinase activity from Candida albicans is thought to be a potential virulence factor. Four laboratories have cloned a gene from C. albicans encoding this enzyme. When two of these genes sharing 77% homology at the DNA level are hybridized under conditions of high stringency to contour-clamped homogeneous electric field chromosome separations of four different strains, they label different chromosomes: chromosome 6 for SAP1 and chromosome R for SAP2. The existence of different genes for the two sequences was confirmed by polymerase chain reaction. Genomic Southern blots probed with the genes and washed at low stringency revealed several cross-hybridizing bands. Contour-clamped homogeneous electric field chromosome separations probed at low stringency indicated that there was a cross-hybridizing sequence on chromosome 3 in addition to those on chromosomes R and 6. The genes for the secreted aspartyl proteinase activity in C. albicans thus constitute a gene family which we have called the SAP family.

Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multigene family with at least nine members (SAP1 to SAP9) and are considered putative virulence factors important for the pathogenicity of this human pathogen. The role of Sap isoenzymes in the virulence of C. albicans has not yet been clearly established, and therefore, using recent progress in the genetics of this yeast, we have constructed a panel of isogenic yeasts, each with a disruption of one or several SAP genes. We focused on the construction of a C. albicans strain in which three related SAP genes (SAP4, SAP5, and SAP6) were disrupted. Growth of the delta sap4,5,6 triple homozygous null mutant DSY459 in complex medium was not affected, whereas, interestingly, growth in a medium containing protein as the sole nitrogen source was severely impaired compared to the growth of the wild-type parent strain SC5314. Since the presence of Sap2 is required for optimal growth on such medium, this suggests that Sap4, Sap5, or Sap6 plays an important role for the process of induction of SAP2. When guinea pigs and mice were injected intravenously with DSY459, their survival time was significantly longer than that of control animals infected with the wild-type SC5314. Attenuated virulence of DSY459 was followed by a significant reduction of yeast cells in infected organs. These data suggest that the group of Sap4, Sap5, and Sap6 isoenzymes is important for the normal progression of systemic infection by C. albicans in animals.

Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.

The endothelial cell interactions of homozygous null mutants of Candida albicans that were deficient in secreted aspartyl proteinase 1 (Sap1), Sap2, or Sap3 were investigated. Only Sap2 was found to contribute to the ability of C. albicans to damage endothelial cells and stimulate them to express E-selectin. None of the Saps studied appears to play a role in C. albicans adherence to endothelial cells.

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wildtype baker's yeast. They were identified by MS and NMR spectroscopy as β-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C26) α-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans as well as a corresponding enzyme from Pichia pastoris.

The gene encoding the Candida albicans aspartate proteinase that is secreted by cells grown in protein-containing media was cloned from a C. albicans genomic bank. The base sequence of the insert shows a 1173 bp open-reading frame and indicates an amino acid sequence typical of aspartate proteinases, with amino acid sequence homology to other enzymes of this class and a putative signal peptide consisting of 50 amino acids upstream of the active enzyme.

The aspartate proteinase inhibitor pepstatin A has been shown previously to reduce the adherence of Candida albicans yeast cells to human surfaces. This suggests that in addition to their presumed function facilitating tissue penetration, the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles as cellular adhesins. We therefore examined the relative adherence of yeast cells of a parental wild-type strain of C. albicans in relation to yeast cells of strains harbouring specific disruptions in various members of the SAP gene family in an otherwise isogenic background. The adhesiveness of Δsap1, Δsap2 and Δsap3 null mutants and a triple Δsap 4–6 disruptant was examined on three surfaces – glass coated with poly-l-lysine or a commercial cell-free basement membrane preparation (Matrigel) and on human buccal epithelial cells. Pepstatin A reduced adherence to all surfaces. Adherence of the each of the single SAP null mutants to these three substrates was either reduced or not affected significantly compared to that of the parental strain. The adherence of the Δsap4–6 mutant was reduced on poly-l-lysine and Matrigel, but increased on buccal cells. The results suggest that in addition to a primary enzymatic role, various SAPs may also act singly or synergistically to enhance the adhesiveness to C. albicans cells to certain human tissues.